1. Field of the Invention
The invention relates generally to compounds and methods for labeling recombinant fusion proteins and in particular to small synthetic molecules that react with target sequences.
2. Background Information
Many techniques in the biological sciences require attachment of labels to macromolecules such as polypeptides. For example, the location of a polypeptide within a cell can be determined by attaching a fluorescent label to the polypeptide.
Traditionally, labeling has been accomplished by chemical modification of purified polypeptides. For example, normal procedures for fluorescent labeling require that the polypeptide be covalently reacted in vitro with a fluorescent dye, then repurified to remove excess dye and/or any damaged polypeptide. Using this approach, problems of labeling stoichiometry and disruption of biological activity are often encountered. Furthermore, to study a chemically modified polypeptide within a cell, microinjection can be required. This can be tedious and cannot be performed on a large population of cells.
A variety of site-specific labeling techniques have been developed and utilized with varying degrees of success, though each technique is not without drawbacks. For example, fusion to Green Fluorescent Protein (GFP) and its different color variants is irreversible and sometimes problematic. Due to the large size of GFP proteins, the fusion can perturb the host protein folding and function. Site-directed mutagenesis incorporating a single cysteine has been used to label proteins, but competing thiols are too ubiquitous in cells to allow labeling in situ. Trivalent arsenic mediated cysteine labeling has been used with some success. However, arsenic only binds reduced thiols and is potentially toxic. Labeling of epitope tags with antibodies has been attempted, but antibodies are large, membrane impermeant, and therefore require microinjection or permeabilization of cells. Single chain antibody-hapten labeling can be utilized, however, antibodies do not fold well in reducing environments. Avidin-biotin labeling suffers from the fact that avidin in the cytosol is either toxic or biotin-saturated. Intein-based labeling systems have not yet been demonstrated in live cells. Finally, the hexahistidine-nickel labeling system is potentially undesirable since nickel is promiscuous, toxic, and quenches fluorescence.
Accordingly, a need exists for improved compositions and methods for labeling polypeptides, especially within or on the surface of living cells.